Method and apparatus for controllably effecting samples using two signals

ABSTRACT

Methods and systems for effecting a parameter and detecting a parameter using two electric signal in a conductive path are described.

[0001] This application is a continuation of U.S. patent applicationSer. No. 10/293,238, filed Nov. 12, 2002, which claims priority fromprovisional patent application 60/332990 filed 13 Nov. 2001, each ofwhich is incorporated herein by reference.

[0002] All documents referenced herein are incorporated in theirentirety by reference for all purposes.

[0003] This application may be related to other patent applications andissued patents assigned to the assignee indicated above. Theseapplications and issued patents are incorporated herein by reference tothe extent allowed under applicable law.

FIELD OF THE INVENTION

[0004] The present invention generally relates to methods and/or systemsfor precisely controlling and measuring heating. More particularly, thepresent invention provides a technique, including methods and devices,for providing and controlling heat to samples in a channel of a microscale sample handling system. Merely by way of example, the invention isapplied to a polymerase chain reaction process, commonly termed PCR,performed in a microfluidic system or device but it will be recognizedthat the invention has a much wider range of applicability. Theinvention according to further embodiments also provides techniques formonitoring and controlling a variety of process parameters usingimpedance and/or conductance measurements.

[0005] According to further embodiments, the invention relates to acomputer method and/or system for precisely determining temperatureand/or controlling heating in specific devices.

BACKGROUND OF THE INVENTION

[0006] The discussion of any work, publications, sales, or activityanywhere in this submission, including in any documents submitted withthis application, shall not be taken as an admission by the inventorsthat any such work constitutes prior art. The discussion of anyactivity, work, or publication herein is not an admission that suchactivity, work, or publication existed or was known in any particularjurisdiction.

[0007] There has been a growing interest in the manufacture and use ofmicroscale systems for the acquisition of chemical and biochemicalinformation. Techniques commonly associated with the semiconductorelectronics industry, such as photolithography, wet chemical etching,etc., are being used in the fabrication of microscale systems, such asmicrofluidic systems. The term “microfluidic” refers generally to asystem or device or “chip” having channels and chambers which aregenerally fabricated at the micron or submicron scale, e.g., having atleast one cross-sectional dimension in the range of from about 0.1 μm toabout 500 μm. Early discussions of the use of planar chip technology forthe fabrication of microfluidic systems are provided in Manz et al.,Trends in Anal. Chem. (1990) 10(5):144-149 and Manz et al., Adv. inChromatog. (1993) 33:1-66, which describe the fabrication of suchfluidic devices and particularly microcapillary devices, in silicon andglass substrates.

[0008] Applications of microscale and/or microfluidic systems aremyriad. For example, International Patent Appln. WO 96/04547, publishedFeb. 15, 1996, describes the use of microfluidic systems for capillaryelectrophoresis, liquid chromatography, flow injection analysis, andchemical reaction and synthesis. U.S. Pat. No. 5,942,443, assigned tothe present assignee, discloses wide ranging applications microfluidicsystems in rapidly assaying large number of compounds for their effectson chemical, and preferably, biochemical systems. The phrase,“biochemical system”, generally refers to a chemical interaction whichinvolves molecules of the type generally found within living organisms.Such interactions include the full range of catabolic and anabolicreactions which occur in living systems including enzymatic, binding,signaling and other reactions. Biochemical systems of particularinterest include, e.g., receptor-ligand interactions, enzyme-substrateinteractions, cellular signaling pathways, genetic analysis, transportreactions involving model barrier systems (e.g., cells or membranefractions) for bio-availability screening, and a variety of othergeneral systems.

[0009] Many chemical or biological systems also benefit from controlover processing parameters such as temperature, concentration ofreagents, buffers, salts and other materials, and the like. Inparticular, some chemical or biological systems require processes to becarried out at controlled and/or controllably varied temperature. Inproviding such a controlled temperature in miniaturized fluidic systems,external heating elements have generally been used. Such heatingelements typically include external resistive heating coils or material,which provide heat to the fluidic system in a conductive manner. Thisheating unit attaches itself directly to an external portion of the chipto globally heat the chip and to provide a uniform temperaturedistribution to be present on the chip. This external heating unit,however, is cumbersome. It also complicates chip manufacturing and oftenaffects quality and reliability of the chip. Additionally, the externalheating element can fail and generally cannot effectively control heatsupplied to the chip, which can cause undesirable temperature gradientsand fluctuations in the chip. Accordingly, the external heating elementapplied to a chip is limited and can be unreliable in controllingprocess temperature in the chip.

[0010] Larger scale temperature controllers have also been used tocontrol reaction temperatures within a reaction vessel, including, e.g.,hot-plates, water baths, and the like. Such controllers are not wellsuited to providing accurate control of temperature within amicrofluidic system. In fact, such global heating systems heat theentire material region of the microfluidic device and cannot be used toselectively apply heat to specific regions of the microfluidic device,e.g., specific channels or chambers. Additionally, these largetemperature controllers, e.g., hot plates, often require large heatingelements, which transfer heat via conduction. These heating elementspossess a large characteristic response time, which often relates to along time to heat or cool material within a reaction vessel in contacttherewith in some applications.

SUMMARY OF THE INVENTION

[0011] Various strategies have been proposed for providing heating in amicroscale (such as microfluidic) device. Among these strategies, threeof particular interest to the present invention are (1) Joule (orelectrolytic) heating, (2) in-channel resistive heating, and (3)proximal resistive heating. In each of these types of heating, anelectric signal is used to provide energy. In Joule heating, theelectric signal is passed directly through the sample to be heated(which generally must be an electrolytic material, thus the alternativename electrolytic heating.) Electrical energy is converted to heat as itpasses through the sample. In resistive heating, a separate conductor(such as a metal or semiconductor channel) is used to carry the electricsignal. The impedance and/or resistance of this separate conductorcauses the conductor to heat due to electric signal flow. This heat isthen transferred by heat conduction to a sample in a microscale devicechannel or region. In in-channel resistive heating, one or more heatingelements is placed in the channel, possibly in contact with the samplematerial. In proximal resistive heating, one or more heating elements isplaced near the channel.

[0012] Various of the above general types of heating strategies has beenproposed using either DC electrical signals or AC electrical signals. Ithas also been proposed to detect effects of heating (such as thetemperature) or other effects using conductance or impedance of theapplied electrical signal. However, there is a continuing need forrefined and improved techniques for effecting and/or detecting heatingor other parameters in microscale devices. A number of earlier patentsdiscuss various aspects related to the operation and/or construction ofmicrofluidic systems. An example of these include U.S. Pat. No.5,965,410 (Electrical current for controlling fluid parameters inmicrochannels); U.S. Pat. No. 5,779,868; U.S. Pat. No. 5,800,690; U.S.Pat. No. 6,306,590; and U.S. Pat. No. 6,171,850 (Integrated devices andsystems for performing temperature controlled reactions and analyses).

[0013] According to the present invention, two different signals areused one signal to provide energy for heating (or, in alternativeembodiments, effecting other parameters) and another signal at adifferent frequency used as a probe to measure the effect on theparameter. The second signal, in specific embodiments, is an electricalsignal having a different frequency than the signal used for heating.Thus, in specific embodiments, the invention provides a mechanism thatallows a detection or probe signal to remain separated from an effectorsignal without requiring physically separate conductive paths.

[0014] Specific embodiments according to the invention include (1) ajoule heating system and/or method wherein the effector signal and probesignal are signals through an electrolytic sample medium (2) anin-channel resistive heating system and/or method wherein the effectorsignal and probe signal are signals through an in-channel electricalconductor; and (3) a proximal resistive heating system and/or methodwherein the effector signal and probe signal are signals through anelectrical conductor proximal to a channel. Specific embodimentsaccording to the invention also include (1) a heating system and/ormethod wherein the effector signal is a DC signal and the probe signalis a distinguishable AC, e.g. of about 10 Hz; and (2) a heating systemand/or method wherein the effector signal is a higher frequency ACsignal and the probe signal is a distinguishable lower frequency ACsignal.

[0015] While various combinations of the above characteristics arepossible, according to specific embodiments of the invention a DCheating signal is generally not used when the signal passes through thesample (joule heating).

[0016] In various embodiments, the present invention can be embodied ina microfluidic system. In particular, such a system comprises asubstrate having at least a first fluid-filled (as used herein, fluidalso includes a gel) microscale channel disposed therein. The systemalso includes a means for generating or receiving the first and secondsignals for controlling temperature of the fluid in the first portion ofthe channel.

[0017] In a related aspect, the present invention provides amicrofluidic system having one or more thermal elements includedtherein. The system comprises a first channel defined in a substrate,where the channel includes a first end and a second end. A first energysource is provided coupled between the first end and the second end ofthe channel. The first energy source is applied such that a portion ofsaid material is heated in a portion of said first capillary channel.

[0018] A second energy source is also provided coupled to the fluid inthe first channel, whereby a signal from the second energy source can bemonitored to measure a characteristic of the channel and/or materialtherein. In further embodiments, this measurement can be used to controladjustment of parameters in the fluid. Typically, a third energy sourcecan also be provided coupled to the channel, whereby a signal from thethird energy source is further set at a current or voltage such that afluid is pumped through said first channel.

[0019] In still another related aspect, the present invention provides amicrofluidic system, that comprises a capillary channel comprising afirst end and a second end, and at least two signal (e.g., energy)sources (or a single source divided or filtered to provide at least twodifferent signals) coupled between the first end and the second end. Thesource(s) provide voltage and/or current signals coupled to the channeland/or material such that a portion of a fluid is heated in a portion ofthe capillary channel using one signal and such that parameters, such astemperature, are measured by a second signal.

[0020] In a further related aspect, the present invention provides amicrofluidic system, which comprises a capillary channel defined in asubstrate, the channel comprising fluid therein, wherein the capillarychannel has a region whereupon fluid in the region is selectively heatedusing a voltage bias applied to the fluid in the capillary channel, withtemperature measured using a second signal applied to the fluid in thecapillary channel.

[0021] In a further related aspect, the present invention also providesa multi-port, microfluidic device, which comprises a substrate having afirst fluid-filled channel region defined therein. The substrateincludes at least a first port and a second port for transporting amaterial therebetween, and a second channel region defined in thesubstrate for applying electric current for heating fluid between thefirst and second ports and for applying a signal for measuringtemperature in the fluid.

[0022] The present invention also provides a computer program productfor operating a microfluidic system in accordance with other aspects ofthe present invention. In particular, the computer program comprises acomputer readable memory including a code that directs an energy sourceto adjust an electric current or voltage to a channel comprising a fluidtherein, to heat the fluid to a selected elevated level and formeasuring temperature using a second applied electrical signal.

[0023] The present invention also provides methods of controllingtemperatures in microfluidic systems as herein described.

[0024] Aspects of one illustrative embodiment of a the invention isdescribed below as it might be implemented on a general purpose computerusing a suitable programming language such as Java, C++, Cobol, C,Pascal, Fortran., PL1, LISP, assembly, etc. In the interest of clarity,not all features of an actual implementation are described in thisspecification. It will be understood that in the development of any suchactual implementation (as in any software development project), numerousimplementation-specific decisions must be made to achieve thedevelopers' specific goals and subgoals, such as compliance with system-and business-related constraints, which will vary from oneimplementation to another. Moreover, it will be appreciated that such adevelopment effort might be complex and time-consuming, but wouldnevertheless be a routine undertaking of software engineering for thoseof ordinary skill having the benefit of this disclosure.

Other Features & Benefits

[0025] The invention and various specific aspects and embodiments willbe better understood with reference to the following drawings anddetailed descriptions. In different figures, similarly numbered itemsare intended to represent similar functions within the scope of theteachings provided herein. In some of the drawings and detaileddescriptions below, the present invention is described in terms of theimportant independent embodiment of a microfluidic device or system.This should not be taken to limit the invention, which, using theteachings provided herein, can be applied to other situations in whichit is desired to control the heating of fluids. For purposes of clarity,this discussion refers to devices, methods, and concepts in terms ofspecific examples. However, the invention and aspects thereof may haveapplications to a variety of types of devices and systems. It istherefore intended that the invention not be limited except as providedin the attached claims.

[0026] Furthermore, it is well known in the art that logic and/orelectronic systems and methods such as described herein can include avariety of different components and different functions in a modularfashion. Different embodiments of the invention can include differentmixtures of elements and functions and may group various functions asparts of various elements. For purposes of clarity, the invention isdescribed in terms of systems that include many different innovativecomponents and innovative combinations of innovative components andknown components. No inference should be taken to limit the invention tocombinations containing all of the innovative components listed in anyillustrative embodiment in this specification. The functional aspects ofthe invention that are implemented on a computer, as will be understoodfrom the teachings herein, may be implemented or accomplished using anyappropriate implementation environment or programming language, such asC, C++, Cobol, Pascal, Java, Java-script, HTML, XML, dHTML, assembly ormachine code programming, etc. All references, publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1 is a simplified schematic illustration of an embodiment ofa microfluidic system according to specific embodiments of the presentinvention.

[0028]FIG. 2 is a simplified schematic illustration of two designsembodiment of a microfluidic device with resistive heating according tospecific embodiments of the present invention.

[0029]FIG. 3 is a block diagram showing components an illustrativesystem embodiment for effecting a parameter (e.g., temperature) in adevice according to specific embodiments of the present invention.

[0030]FIG. 4A is a block diagram showing components of a more detailedexample system embodiment for controlling Joule heating in a microscaledevice according to specific embodiments of the present invention.

[0031]FIG. 4B is a block diagram showing components of an example systemembodiment for controlling proximal channel heating in a microscaledevice according to specific embodiments of the present invention.

[0032]FIG. 5A is a block diagram illustrating a configuration forproviding heating in a channel with a central signal electrode and twoground electrodes according to specific embodiments of the presentinvention.

[0033]FIG. 5B is a block diagram illustrating a configuration forproviding heating in a channel with a three separate control loopsaccording to specific embodiments of the present invention.

[0034]FIG. 5C is a block diagram illustrating a configuration forproviding heating in a channel with some modifications from the systemshown in 4A according to specific embodiments of the present invention.

[0035]FIG. 6 is a block diagram illustrating software modules that canbe used as a controller to operate an AC heater according to specificembodiments of the present invention.

[0036]FIG. 7 is a simplified diagram of a microfluidic system with aheating source according to the present invention.

[0037]FIG. 8 is a simplified diagram of a microfluidic system with aheating source according to an alternative embodiment of the presentinvention.

[0038]FIG. 9 is a block diagram showing a representative example logicdevice in which various aspects of the present invention may beembodied.

DETAILED DESCRIPTION OF THE INVENTION

[0039] 1. Glossary

[0040] Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular compositionsor systems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, the singular forms “a”, “an” and“the” include plural referents unless the content and context clearlydictates otherwise. Thus, for example, reference to “a device” includesa combination of two or more such devices, and the like.

[0041] Unless defined otherwise, technical and scientific terms usedherein have meanings as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inpractice or for testing of the present invention, the preferredmaterials and methods are described herein.

[0042] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0043] The term “microfluidic device” as used herein refers to a devicefor performing nanoliter-scale reactions, typically containing one ormore microscale channels. The term “microscale channel”, or“microchannel,” refers to one or more fluid passages, chambers,conduits, cavities, channels, channel networks, or reservoirs which haveat least one internal cross-sectional dimension, e.g., depth, width,length, diameter, generally for example in the range of about 0.1 micrometers to about 500 micro meters.

[0044] As used herein, the terms “microscale” or “microfabricated”generally refers to structural elements or features of a device whichhave at least one fabricated dimension in the range of from about 0.1micro meters to about 500 micro meters. Thus, a device referred to asbeing microfabricated or microscale will include at least one structuralelement or feature having such a dimension. When used to describe afluidic element, such as a passage, chamber or conduit, the terms“microscale”, “microfabricated” or “microfluidic” generally refer to oneor more fluid passages, chambers or conduits which have at least oneinternal cross-sectional dimension, e.g., depth, width, length,diameter, etc., that is less than 500 μm, and typically between about0.1 μm and about 500 μm. In the devices of the present invention, themicroscale channels or chambers preferably have at least onecross-sectional dimension between about 0.1 μm and 200 μm, morepreferably between about 0.1 μm and 100 μm, and often between about 0.1μm and 20 μm.

[0045] 2. Microfluidic Systems

[0046] Microfluidic devices or systems prepared in accordance with thepresent invention typically include at least one microscale channel,usually at least two intersecting microscale channels, and often, threeor more intersecting channels disposed within a single body structure.Channel intersections may exist in a number of formats, including crossintersections, “T” intersections, or any number of other structureswhereby two channels are in fluid communication. Body structures may beintegrated structures, or may be aggregations of multiple separate partsthat fit together to form the aggregate body structure.

[0047] Typically, the body structure of the microfluidic devicesdescribed herein comprises an aggregation of two or more separate layerswhich when appropriately mated or joined together, form the microfluidicdevice of the invention, e.g., containing the channels and/or chambersdescribed herein. Typically, the microfluidic devices described hereinwill comprise a top portion, a bottom portion, and an interior portion,wherein the interior portion substantially defines the channels andchambers of the device.

[0048]FIG. 1 illustrates a two-layer body structure 10, for amicrofluidic device. In preferred aspects, the bottom portion of thedevice 12 comprises a solid substrate that is substantially planar instructure, and which has at least one substantially flat upper surface14. A variety of substrate materials may be employed as the bottomportion. Typically, because the devices are microfabricated, substratematerials will be selected based upon their compatibility with knownmicrofabrication techniques, e.g., photolithography, wet chemicaletching, laser ablation, air abrasion techniques, injection molding,embossing, and other techniques. The substrate materials are alsogenerally selected for their compatibility with the full range ofconditions to which the microfluidic devices may be exposed, includingextremes of pH, temperature, salt concentration, and application ofelectric fields. Accordingly, in some preferred aspects, the substratematerial may include materials normally associated with thesemiconductor industry in which such microfabrication techniques areregularly employed, including, e.g., silica based substrates, such asglass, quartz, silicon or polysilicon, as well as other substratematerials, such as gallium arsenide and the like. In the case ofsemiconductive materials, it will often be desirable to provide aninsulating coating or layer, e.g., silicon oxide, over the substratematerial, and particularly in those applications where electric fieldsare to be applied to the device or its contents. Although preferredsubstrates are planar in structure, it will be appreciated that avariety of substrate conformations may be utilized, including concave orconvex structures, tubular structures, e.g., capillaries, and the like.

[0049] In additional preferred aspects, the substrate materials willcomprise polymeric materials, e.g., plastics, such aspolymethylmethacrylate (PMMA), polycarbonate, polytetrafluoroethylene(TEFLON™), polyvinylchloride (PVC), polydimethylsiloxane (PDMS),polysulfone, and the like. Such polymeric substrates are readilymanufactured using available microfabrication techniques, as describedabove, or from microfabricated masters, using well known moldingtechniques, such as injection molding, embossing or stamping, or bypolymerizing the polymeric precursor material within the mold (See U.S.Pat. No. 5,512,131). Such polymeric substrate materials are preferredfor their ease of manufacture, low cost and disposability, as well astheir general inertness to most extreme reaction conditions. Again,these polymeric materials may include treated surfaces, e.g.,derivatized or coated surfaces, to enhance their utility in themicrofluidic system, e.g., provide enhanced fluid direction, e.g., asdescribed in U.S. Pat. No. 5,885,470, and which is incorporated hereinby reference in its entirety for all purposes. Further, such alternatesubstrates may be in any of a variety of conformations, e.g., planar,tubular, concave, convex, or the like.

[0050] The channels and/or chambers of the microfluidic devices aretypically fabricated into the upper surface of the bottom substrate orportion 12, as microscale grooves or indentations 16, using the abovedescribed microfabrication techniques. In specific embodiments accordingto the present invention using resistive heating, grooves orindentations 16 can be associated with in-channel or proximal heatingelements using microfabrication techniques.

[0051] The top portion or substrate 18 also comprises a first planarsurface 20, and a second surface 22 opposite the first planar surface20. In the microfluidic devices prepared in accordance with methodsdescribed herein, the top portion also includes a plurality ofapertures, holes or ports 24 disposed therethrough, e.g., from the firstplanar surface 20 to the second surface 22 opposite the first planarsurface.

[0052] The first planar surface 20 of the top substrate 18 is thenmated, e.g., placed into contact with, and bonded to the planar surface14 of the bottom substrate 12, covering and sealing the grooves and/orindentations 16 in the surface of the bottom substrate, to form thechannels and/or chambers (i.e., the interior portion) of the device atthe interface of these two components. The holes 24 in the top portionof the device are oriented such that they are in communication with atleast one of the channels and/or chambers formed in the interior portionof the device from the grooves or indentations in the bottom substrate.In the completed device, these holes function as reservoirs forfacilitating fluid or material introduction into the channels orchambers of the interior portion of the device, as well as providingports at which electrodes may be placed into contact with fluids withinthe device, allowing application of electric fields along the channelsof the device to control and direct fluid transport within the device.

[0053] It will be further understood that microfabrication techniquescan include fabrication of electrical conducting paths either in thechannels or proximal to the channels. These electrical conducting pathscan comprise metals and/or other conducting and/or semiconductingmaterials.

[0054] In some embodiments, the microfluidic devices will include anoptical detection window disposed across one or more channels and/orchambers of the device. Optical detection windows are typicallytransparent such that they are capable of transmitting an optical signalfrom the channel/chamber over which they are disposed. Optical detectionwindows may merely be a region of a transparent cover layer, e.g., wherethe cover layer is glass or quartz, or a transparent polymer material,e.g., PMMA, polycarbonate, etc. Alternatively, where opaque substratesare used in manufacturing the devices, transparent detection windowsfabricated from the above materials may be separately manufactured intothe device.

[0055] These devices may be used in a variety of applications,including, e.g., the performance of high throughput screening assays indrug discovery, immunoassays, diagnostics, genetic analysis, and thelike. As such, the devices described herein, will often include multiplesample introduction ports or reservoirs, for the parallel or serialintroduction and analysis of multiple samples. Alternatively, thesedevices may be coupled to a sample introduction port, e.g., a pipettor,which serially introduces multiple samples into the device for analysis.Examples of such sample introduction systems are described in e.g., U.S.Pat. Nos. 5,880,071 and 6,046,056 each of which is hereby incorporatedby reference in its entirety for all purposes.

[0056] In further embodiments, the devices, methods and systemsdescribed herein, can employ electrokinetic material transport systems,and preferably, controlled electrokinetic material transport systems. Asused herein, “electrokinetic material transport systems” include systemswhich transport and direct materials within an interconnected channeland/or chamber containing structure, through the application ofelectrical fields to the materials, thereby causing material movementthrough and among the channel and/or chambers.

[0057] Although the preferred aspects of the present invention generallyemploy electrokinetic transport of materials in microfluidic systems, itis easily recognized that the heating and control aspects of the presentinvention are readily adaptable to systems utilizing other materialtransport systems. For example, pressure based or pneumatic flow systemsusing pumps and/or pressure sources external to the microfluidic devicecan be used in conjunction with the heating, sensing and control aspectsof the present invention. Similarly, integrated microfluidic devices,e.g., incorporating microfabricated pump and valve structures,integrated into the device, are also readily adaptable for use withthese heating and control systems.

[0058] Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. Other electrokinetic material direction and transportsystems rely upon the electroosmotic flow of fluid and material within achannel or chamber structure, which results from the application of anelectric field across such structures. In brief, when a fluid is placedinto a channel which has a surface bearing charged functional groups,e.g., hydroxyl groups in etched glass channels or glassmicrocapillaries, those groups can ionize. In the case of hydroxylfunctional groups, this ionization, e.g., at neutral pH, results in therelease of protons from the surface and into the fluid, creating aconcentration of protons at near the fluid/surface interface, or apositively charged sheath surrounding the bulk fluid in the channel.Application of a voltage gradient across the length of the channel willcause the proton sheath to move in the direction of the voltage drop,i.e., toward the negative electrode, pulling the bulk fluid along withit.

[0059] “Controlled electrokinetic material transport and direction”, asused herein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547, to Ramsey, whichis incorporated herein by reference in its entirety for all purposes. Inparticular, the preferred microfluidic devices and systems describedherein, include a body structure which includes at least twointersecting channels or fluid conduits, e.g., interconnected, enclosedchambers, which channels include at least three unintersected termini.The intersection of two channels refers to a point at which two or morechannels are in fluid communication with each other, and encompasses “T”intersections, cross intersections, “wagon wheel” intersections ofmultiple channels, or any other channel geometry where two or morechannels are in such fluid communication. An unintersected terminus of achannel is a point at which a channel terminates not as a result of thatchannel's intersection with another channel, e.g., a “T” intersection.In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

[0060]FIG. 2 is a simplified schematic illustration of two designsembodiment of a microfluidic device with resistive heating according tospecific embodiments of the present invention. These designs areprovided as examples, and many other configurations are possibleaccording to specific embodiments of the present invention. In thisfigure, and indicate deep and shallow channel regions, respectively, andindicates metal deposited by microfabrication techniques. In (A) aproximal heating design is shown, where the heating elements areadjacent to the channel (see inset). An in-channel design is shown in(B), with the inset illustrating the metal strip located within thechannel.

[0061] A variety of other aspects of design and/or operation ofcontrolled electrokinetic material transport systems are described inthe references cited herein and these aspects can be used with thesystems, devices, and/or methods of the present invention in specificembodiments. Further, although the devices and systems specificallyillustrated herein are generally described in terms of the performanceof a few or one particular operation, it will be readily appreciatedfrom this disclosure that the flexibility of these systems permits easyintegration of additional operations into these devices. For example,the devices and systems described will optionally include structures,reagents and systems for performing virtually any number of operationsboth upstream and downstream from the operations specifically describedherein. Such upstream operations include sample handling and preparationoperations, e.g., cell separation, extraction, purification,amplification, cellular activation, labeling reactions, dilution,aliquoting, and the like. Similarly, downstream operations may includesimilar operations, including, e.g., separation of sample components,labeling of components, assays and detection operations. Assay anddetection operations include without limitation, probe interrogationassays, e.g., nucleic acid hybridization assays utilizing individualprobes, free or tethered within the channels or chambers of the deviceand/or probe arrays having large numbers of different, discretelypositioned probes, receptor/ligand assays, immunoassays, and the like.

[0062] A variety of controlling instrumentation may be utilized inconjunction with the microfluidic devices described above, forcontrolling the transport and direction of fluids and/or materialswithin the devices of the present invention. For example, in many cases,fluid transport and direction may be controlled in whole or in part,using pressure based flow systems that incorporate external or internalpressure sources to drive fluid flow. Internal sources includemicrofabricated pumps, e.g., diaphragm pumps, thermal pumps, lamb wavepumps and the like that have been described in the art. See, e.g., U.S.Pat. Nos. 5,271,724, 5,277,556, and 5,375,979 and Published PCTApplication Nos. WO 94/05414 and WO 97/02357. In such systems, fluiddirection is often accomplished through the incorporation ofmicrofabricated valves, which restrict fluid flow in a controllablemanner. See, e.g., U.S. Pat. No. 5,171,132.

[0063] One or more aspects of systems described herein generally includecontroller systems for use in conjunction with the microfluidic devicestypically include an electrical power supply and circuitry forconcurrently delivering appropriate voltages to a plurality ofelectrodes that are placed in electrical contact with the fluidscontained within the microfluidic devices or appropriate voltagesapplied to conductive paths proximal to fluids contained within thedevices. Examples of electrical controllers include those described in,e.g., U.S. Pat. No. 5,965,001 and International Patent Publication No.WO 98/00707, the disclosures of which are hereby incorporated herein byreference in their entirety for all purposes. In brief, the controlleruses electric current control in the microfluidic system. The electricalcurrent flow at a given electrode is directly related to the ionic flowalong the channel(s) connecting the reservoir in which the electrode isplaced. This is in contrast to the requirement of determining voltagesat various nodes along the channel in a voltage control system. Thus thevoltages at the electrodes of the microfluidic system are set responsiveto the electric currents flowing through the various electrodes of thesystem.

[0064] 3. Illustrative System Architecture

[0065]FIG. 3 is a block diagram showing components an illustrativesystem embodiment for effecting a parameter (e.g., temperature) in adevice according to specific embodiments of the present invention.According to the present invention, two different signals are used, onesignal to provide energy for effecting parameters of the sample (e.g.,heating, causing an ionizing reaction, moving cells or other material ina sample,etc.) and another signal at a different frequency used as aprobe to measure a conductance parameter and to provide feedback tomodify the first signal. In specific embodiments, these signals can beused in the same conductive path. Thus, in specific embodiments, theinvention provides a mechanism that allows a detection or probe signalto remain separated from an effector signal without requiring physicallyseparate conductive paths.

[0066] Specific embodiments generally illustrated by FIG. 2 include (1)a Joule heating system and/or method wherein the effector signal andprobe signal are signals through an electrolytic sample medium (2) anin-channel resistive heating system and/or method wherein the effectorsignal and probe signal are signals through an in-channel electricalconductor; (3) a proximal resistive heating system and/or method whereinthe effector signal and probe signal are signals through an electricalconductor proximal to a channel, (4) a cell transport system and/ormethod wherein the effector signal and probe signal are signals througha cell sample medium. Specific embodiments according to the inventionalso include (1) a heating system and/or method wherein the effectorsignal is a DC signal and the probe signal is a distinguishable AC, e.g.of about 10 Hz; and (2) a heating system and/or method wherein theeffector signal is a higher frequency AC signal and the probe signal isa distinguishable lower frequency AC signal.

[0067] In an example embodiment, a system according to specificembodiments of the present invention can be understood as included fourchief components: (1) a LabChip® 10 or similar device that holds afluidic or other material that is affected and probed by electricalsignals including at least one conductive path for a probe signal and aneffector signal; (2) a power source 40 for supplying one or moreeffector electrical signals that alter parameters at device 10; (4) aprobe 60 including a probe signal generator 62 for generating a probeelectric signal different than the effector signals and a probe detector64 for detecting a probe electric signal; and (5) a controller 80 toclose a control loop between probe 60 and the controllable power source40.

[0068] Sample Device 10

[0069] In particular embodiments, device 10 can be understood as anymicroscale and/or microfluidic and/or analogous device. As will beunderstood from the references and discussions herein, such a deviceincludes at least one appropriate conductive path, which in specificembodiments can be either through a sample, in-channel with a sample, orproximal to a sample channel. Methods and systems of according tospecific embodiments of the present invention can be used with anysample container, including larger scale samples.

[0070] Effector Power Source

[0071] Effector power source 40 can comprise any appropriate circuit ordevice for controllably applying appropriate power levels to have aneffect on a sample. It is well-known in the art that all electroniccircuits require some source of electric power. Such source can includebattery power, locally generated power, or power from a public utilityor private utility power grid. In the art of electronic systems,typically a variety of different circuits will be used to generate adesired power signal from an easily available electrical power source(such as from a battery, or 60 Hz, 3-phase 110 volt AC power, or 220volt power). Many electronic systems include a power supply that forexample, inputs 110 volt and/or 220 volt AC power and that converts thatpower to various different DC voltages for driving the components of anelectronic circuit and these can be used according to specificembodiments of the present invention.

[0072] In the present invention, in specific embodiments, effector powersource 40 refers to any device that is capable of using an availableelectrical power source and is capable of controllably outputting aneffector signal to device 10. For embodiments using DC current, such apower source can be any known electrical circuit configuration forproviding a voltage and/or current source that is controllable. In asimple embodiment, control can include simply switching off a powersource. In other embodiments, signal power source 40 is a power sourcethat can provide a voltage signal with at least one variable parameter,such as, for example a voltage source with a voltage amplitude that canbe varied from about 80 volts down to about 0 volts for resistiveheating systems or a voltage source that can be varied from about 3800volts to about 0 volts for through the sample effects, e.g. Jouleheating.

[0073] For embodiments using an AC effector signal, effector powersource 40 includes some type of frequency generator, also generally witha variable voltage amplitude and possibly with variable current and/orfrequency output values.

[0074] Probe

[0075] Probe 60 comprises a signal source 62 of a particular frequency fand a detector 64 for measuring conductance at the device using thesignal source. Many different circuit configurations are known in theart for performing this function. As one example, a number of differentoff-the-shelf “lock-in amplifier” type circuit devices are availablethat can be programmed to output a frequency and to measure an impedancedetected as that frequency. Depending on the configuration, thedetection part of probe 60 can include a by-pass filter as known in theart for diverting power at non-detected frequencies away from thedetector.

[0076] In specific embodiments, probe 60 can also comprise a spectrumanalyzer type device that is able to measure conductance/impedanceparameters at a number of different frequencies or a range offrequencies.

[0077] Controller

[0078] Controller 80 receives output data and/or signal from probe 60and provides the control function in a feed-back loop to change theeffector signal to maintain the desired conductance. A variety ofcontroller feed-backs can be used according to specific embodiments ofthe present invention, with the complexity determined by specificapplications and the desired flexibility of a system. According tospecific embodiments of the present invention, controller 80 comprisesanalog circuits for connecting an output signal from probe 60 to acontrol input signal of power source 40. In alternative embodiments,controller 80 comprises a programmable and/or digital controller forproviding such a feedback and possibly for setting other parameters.

[0079] As will be further understood from the following discussion,electrical connections to lab chip 10 of FIG. 3 can be made in variousways. In one embodiment, the applied signals are applied and thedetection signal is received over a single connection to device 10 asillustrated in FIG. 3. In alternative embodiments, power source 10and/or probe signal source 62 and/or probe signal source 64 can beconnected at different parts of the conductive path, as furtherdescribed below.

[0080] 4. Example Detailed System Architecture for Dual-Signal Control

[0081]FIG. 4A is a block diagram showing components of a more detailedexample system embodiment for controlling Joule heating in a microscaledevice according to specific embodiments of the present invention. Thisfigure illustrates an implementation using available off-the-shelfcomponents for system components, operated and modified as hereindescribed.

[0082] In the embodiment shown in FIG. 4A, the sample device isrepresented by a LabChip™ 10 that holds a fluid or other material whichis manipulated and probed by electrical signals. Effector power source40 in this embodiment is an SRS DS345 Synthesized Function Generator.Such a generator is an off-the-shelf device well known in the art andincludes a power supply and inputs for controlling the frequency andamplitude of an output signal. In the embodiment shown, an AT 1502 audioamplifier is used to step up the SRS DS345 voltage signal, for exampleby a factor of 28. A transformer 44 is further used to increase thevoltage sufficiently to achieve a 3800 V_(rms) output desirable forJoule heating embodiments. In resistive heating embodiments, such atransformer generally would not be needed as the maximum voltage outputof power source 40 is about 80-100 volts. Operating specifications shownin the figure are given as examples of specific embodiments of thepresent invention and are not intended to be limiting.

[0083] In a specific example embodiment, probe 60 further comprises anSRS SR830 DSP lock-in amplifier 62. This circuit is well known in theart and can be set to provide a desired probe output frequency f (e.g.,10 Hz) at a given voltage. As known in the art, this circuit provides aseparate output indicating the output current, which indicates theconductance seen at the output frequency.

[0084] The detection circuit, according to specific embodiments of thepresent invention, further comprises a multi-stage low-pass filter 64.In this example, this filter comprises capacitances 64 and impedances66. An A/I (amplifier input) 68 (float) 1 k Ohm impedence to VirtualGround is provided to detect the probe signal that passes through thechannel. While these elements are shown as passive capacitors andresistors, it will be understood that the elements are functionaldesignation, so in some embodiments they may be replaced by circuitelements like transistors that perform the same function.

[0085] In this example embodiment, controller 80 further comprises asoftware PID controller (such as LabVIEW) to close the loop. Thiscontroller receives a data signal from comductance probe 60 and fromthat signal determines the desired control signal to output to powersource 40.

[0086]FIG. 4B is a block diagram showing components of an example systemembodiment for controlling proximal channel heating in a microscaledevice according to specific embodiments of the present invention. Inthis figure, because a lower power signal may be used for heating(including a DC signal) the circuit may be simplified in that some ofthe filtering capacitances and transformer elements are not needed.

[0087] 5. Example Channel Construction with Single-loop or Multi-LoopControl

[0088]FIG. 5A is a block diagram illustrating a configuration forproviding heating in a channel with a central signal electrode and twoground electrodes according to specific embodiments of the presentinvention. As can be seen in this example, both a heating signal (e.g.,about 8 kHz) and a probe signal (in this example of 10 Hz) can beapplied to an electrode 120 in contact with a channel. Both of thesesignals go to ground through a first electrode 122 a and an optionalsecond electrode 122 b. However, the heating signal is passed directlyto ground through a filter (in this example, a capacitor 126). The probefrequency enters a demodulated current sensor 128, where current ismeasured. As will be understood from the teachings herein, a controlloop (not shown) can then be used from the current sensor to controlapplication of the heating signal. A by-pass capacitor is shown in thisexample around the probe signal generator for isolating the probe signalgenerator from the heating signal. In various embodiments of theinvention, a system and/or method according to the invention can involvea low frequency signal in a range of about 5-15 Hz and a high frequencysignal of about 5-15 kHz.

[0089]FIG. 5B is a block diagram illustrating a configuration forproviding heating in a channel with a three separate control loopsaccording to specific embodiments of the present invention.

[0090] In this embodiment, effectively three control loops are used tocontrol the three heating signal sources, to establish three heatingregions in the channel.

[0091] 6. Using AC Signals for In-Channel Resistive Heating

[0092] According to specific embodiments of the present invention, afirst AC signal is used for in-channel resistive heating with a secondAC signal used for probing heat achieved. Using such a system allowsoptimum selection of the AC frequency to keep the signal in theconductive path without substantially entering the sample fluid.

[0093] 7. Example Controller

[0094] According to various specific embodiments of the presentinvention, a controller uses the detected probe signal parameter tocontrol the effector signal(s). As an example, software written inLabVIEW can be used to control the temperature (and, optionally furtherto acquire fluorescence data from the microscope). The temperature wascontrolled with a PID subroutine package. FIG. 6 is a flow diagramillustrating how the LabVIEW subVIs (LabVIEW programs are referred to asVIs for virtual instruments, and subVIs are subroutines.) are organizedand communicate to operate the AC heater. The arrows representcommunication links to the various other subVIs; their size and shadingare indicators of how fast they talk to each other. According tospecific embodiments of the present invention, the functions of eachsubVI can be generally understood as follows:

[0095] Global Variables and Parameters.vi

[0096] This is a place where all the variables that describe the system(experimental parameters) and where all the data that is changing on acontinual basis that needs to be shared between subVIs (livingvariables) are stored.

[0097] The Experimental Parameters are:

[0098] DAQ Device

[0099] ADC Channels

[0100] DAC Output Channel

[0101] DAC Shutter Channel

[0102] Lock-In Sensitivity

[0103] Function Generator Amplitude (V_(rms))

[0104] Reference Temperature (° C.)

[0105] Reference Conductivity (° C.)

[0106] Filename

[0107] Comments

[0108] The Living Variables are:

[0109] Temperature Setpoint (° C.)

[0110] Lock-In Voltage Setpoint

[0111] Lock-In Voltage Output

[0112] % Output

[0113] Binary Data (data read from ADC inputs with 0-2¹² (4096) fullscale)

[0114] Cycle#

[0115] Status Cluster (Temperature, Time, and Shutter State)

[0116] Read Data & Control Temperature.vi

[0117] This module reads in the data/voltages from the PMT(s) (PhotoMultiplier Tubes for detecting fluorescence) and the Lock-In amplifierand updates the data in the living variables section of Global Variablesand Parameters.vi. In addition, this program controls the temperature bychanging the output from the function generator. The % Output is thepercentage of the amplitude displayed on the front panel of the functiongenerator that is going into the amplifier/transformer circuit.

[0118] Live Data Viewer.vi

[0119] This program can be used to continually display data such as:temperature, temperature setpoint, % output, and the fluorescence signalread on channels A and B. It obtains the data from the Global Variablesand Parameters.vi at the user specified rate. When the program stops, itprompts the user to save the data to a file.

[0120] PCR Temperature Profiler.vi

[0121] This program changes the temperature setpoint (and voltagesetpoint) in Global variables and Parameters.vi to generate thetemperature profile inputted by the user. The user selects a state,which consists of a temperature setpoint, hold time, and a shutter state(open or closed). States can be chosen for a hard melt, final extension,and a series of states which can be programmed to cycle through anynumber of times. When the cycling is completed, the program changes thetemperature setpoint to 20° C. to leave the system in a safe, lowvoltage state.

[0122] Shutter.vi

[0123] Toggles the shutter state by changing the voltage on the DAC outpin. There are two versions of this subVI, one that the PCR TemperatureProfiler calls, “Shutter (background).vi,” and one that the operatoruses to manually open and close the shutter, “Shutter.vi.” The user onlyneeds to open “Shutter.vi.”

[0124] Data Reviewer.vi

[0125] Opens data from the designated file and plots it up in the sameformat as in the Live Data Viewer.vi. This is not essential foroperation, but is a convenient feature when you want to revisit olddata.

[0126] According to specific embodiments of the present invention, DataReviewer.vi and Shutter.vi are independent of the other subVIs. Thus,only the programs that are enclosed by heavy lines need to be open whenusing the heater for PCR on a chip.

[0127] 8. Other Example Microfluidic System Characteristics

[0128] Many variations in the overall design of microscale systems arepossible. It will be understood from the teachings herein that heatingand controlling according to specific embodiments of the presentinvention can be incorporated into such variations. Details ofvariations in microscale systems are discussed in the cited references.Some characteristics of various microscale system are discussed furtherbelow.

[0129] Example General Detection Methods

[0130] In the microfluidic systems described herein, a variety ofdetection methods and systems may be employed, depending upon thespecific operation that is being performed by the system. Often, amicrofluidic system will employ multiple different detection systems formonitoring the output of the system. Examples of detection systemsinclude optical sensors, temperature sensors, pressure sensors, pHsensors, conductivity sensors, and the like. Each of these types ofsensors is readily incorporated into the microfluidic systems describedherein. In these systems, such detectors are placed either within oradjacent to the microfluidic device or one or more channels, chambers orconduits of the device, such that the detector is within sensorycommunication with the device, channel, or chamber. The phrase “withinsensory communication” of a particular region or element, as usedherein, generally refers to the placement of the detector in a positionsuch that the detector is capable of detecting the property of themicrofluidic device, a portion of the microfluidic device, or thecontents of a portion of the microfluidic device, for which thatdetector was intended. For example, a pH sensor placed in sensorycommunication with a microscale channel is capable of determining the pHof a fluid disposed in that channel. Similarly, a temperature sensorplaced in sensory communication with the body of a microfluidic deviceis capable of determining the temperature of the device itself.

[0131] The detector may exist as a separate unit, but is preferablyintegrated with the controller system, into a single instrument.Integration of these functions into a single unit facilitates connectionof these instruments with the computer (described below), by permittingthe use of few or a single communication port(s) for transmittinginformation between the controller, the detector and the computer. Asnoted above, and as described in greater detail below, either or both ofthe controller system and/or the detection system are coupled to anappropriately programmed processor or computer which functions toinstruct the operation of these instruments in accordance withpreprogrammed or user input instructions, receive data and informationfrom these instruments, and interpret, manipulate and report thisinformation to the user. As such, the computer is typicallyappropriately coupled to one or both of these instruments (e.g.,including an AD/DA converter as needed).

[0132] The computer typically includes appropriate software forreceiving user instructions, either in the form of user input into a setparameter fields, e.g., in a GUI, or in the form of preprogrammedinstructions, e.g., preprogrammed for a variety of different specificoperations. The software then converts these instructions to appropriatelanguage for instructing the operation of the fluid direction andtransport controller to carry out the desired operation. The computerthen receives the data from the one or more sensors/detectors includedwithin the system, and interprets the data, either provides it in a userunderstood format, or uses that data to initiate further controllerinstructions, in accordance with the programming, e.g., such as inmonitoring and control of flow rates, temperatures, applied voltages,and the like.

[0133] Temperature Control in Microfluidic Systems

[0134] As noted previously, the present invention in particularembodiments involves microfluidic systems that selectively provideenergy to heat materials, e.g., fluids, including samples, analytes,buffers and reagents, in a desired location(s), e.g., within selectedchannels and/or chambers, of the microfluidic device in an efficientmanner. In particular, the present invention uses power source(s) thatpasses electrical current through fluid that is disposed within thechannels and/or chambers of microfluidic systems, for heating thatmaterial in a controlled manner. The present invention, therefore, takesthe art recognized problem of resistive electrical heating of fluids inelectrically controlled systems, and utilizes it to the advantage of theexperimenter, e.g., to perform heating and control operations, withinmicrofluidic systems.

[0135] To provide more precise control of heating, including allowingfor more rapid heating, in further embodiments, the invention involvesthe use of a second probe electrical signal to detect temperaturecharacteristics of a sample. This second signal, in specificembodiments, is an electrical signal having a different frequency thanthe signal used for heating. Thus, in specific embodiments, theinvention provides a mechanism that allows a detection or probe signalto remain separated from an effector signal without requiring physicalseparation.

[0136] The methods and systems of the present invention provide amultitude of advantages over typical temperature control methods forfluidic systems. For example, such systems provide an ease of controland automation that come with precise electrical control of thetemperature. Further such systems provide advantages of speed inchanging temperatures of fluids and materials within channels.Additionally, these systems are readily integrated into state of the artelectrokinetic microfluidic systems. Finally, such methods and systemspermit the precise regional control of temperature control and/orheating within separate microfluidic elements of a given device, e.g.,within one or several separate channels in a given device, withoutheating other regions where such heating is less desired. In particular,in accordance with the presently described methods and systems, heat isonly generated within the fluidic elements where such heating isdesired. Further, because such microfluidic elements are extremely smallin comparison to the mass of the substrate in which they are fabricated,such heat remains substantially localized, e.g., it dissipates into andfrom the substrate before it affects other fluidic elements. In otherwords, the relatively massive substrate functions as a heat sink for theseparate fluidic elements contained therein. Thus, in accordance withthe present invention, one can selectively heat materials in one or morechannels of an integrated microfluidic channel system, e.g., havingmultiple intersecting channels, or multiple channels that are closelypacked together on a single substrate or body structure, while notsubstantially altering the temperature of material in other channels onthe substrate or intersecting with the heated channel.

[0137] In some of the embodiments, a portion of the power goes intokinetic energy of moving the fluid through the channel and a selectedportion of the power to heat the fluid in the channel or a selectedchannel region(s). This channel region may be narrower or smaller incross-section than other channel regions in the channel structure. Thesmaller cross-section provides higher resistance in the fluid, whichincreases the temperature of the fluid as electric current passestherethrough. Alternatively, the electric current can be increased alongthe length of the channel by increased voltage, which also increases theamount of power dissipated into the fluid to correspondingly increasefluid temperature.

[0138]FIG. 7 is a simplified diagram of one example of a microfluidicsystem 400 with a heating source according to the present invention. Thediagram is merely an illustration and should not limit the scope of theclaims herein. One of ordinary skill in the art would recognize othervariations, alternatives, and modifications. The diagram illustrates achannel network or configuration 405 for moving and heating a volume ofmaterial, e.g., fluid, in channel or capillary region 407, which islocated at the intersection of channels 413 and 421. Channel 413 canhave a similar length and width such as those described herein, but canalso be others. Channel 413 connects to regions 409 and 411. Regions 409and 411 supply power to channel 413 for moving the fluid between regions409 and 411. Material is moved between these regions by electrokineticeffects, e.g., electroosmotic and/or electrophoretic forces, such asthose previously described herein, but is not limited to these effects.Power supply 403 provides power through lines 415 and 417 to regions 409and 411, respectively. In particular, power supply 403 provides avoltage differential or electric field between regions 409 and 411 byapplication of a voltage differential to electrodes in regions 409 and411, which voltage differential drives the electrokinetic movement ofthe material. As shown, the voltage differential applies along thelength of channel 413.

[0139] Preferably, power supply 403 also provides power to regions 423and 425 for the purpose of heating the fluid and material in region 407in the channel configuration. In particular, power supply 403 provides avoltage differential between regions 423 and 425, resulting in anelectric current between regions 423 and 425. The electric current isused to distribute energy to the fluid and material in region 407 for atleast heating purposes. Channel 421 includes a novel geometricconfiguration, which is designed to effectively heat the fluid in region407 in an efficient manner. As shown, channel 421 includes outerportions 421, each having a larger width or cross-sectional (e.g.,diameter) dimension than inner portion or region 407, which has acorresponding larger fluid or electrical resistance than outer portions421. The precise dimensions of the wider and narrower portions can beoptimized depending upon the amount of current applied through thesystem, the amount of desired heating, the thermal capacity of thesubstrates and the like, which can be easily optimized experimentally.In any event, such dimensions typically fall within the dimensionsdescribed for microscale channels, herein, e.g., at least onecross-sectional dimension between 0.1 and 500 μm.

[0140] To selectively control the temperature of fluid or material atregion 407 of the channel, power supply 403 applies voltage and/orcurrent in one of many ways. For instance, power supply 403 appliesdirect current (i.e., DC), which passes through channel 421 and intochannel region 407 which is smaller in cross-section to heat fluid andmaterial in region 407. This direct current can be selectively adjustedin magnitude to complement any voltage or electric field that may beapplied between regions 409 and 411 to move materials in and out ofregion 407. In order to heat the material within region 407, withoutadversely affecting the movement of that material, alternating current(i.e., AC) can be selectively applied by the power supply 403 throughchannel 421 and into channel region 407 to heat fluid in region 407.This alternating current used to heat the fluid can be selectivelyadjusted to complement voltage or electric field that may be appliedbetween regions 409 and 411 to move fluid in and out of region 407. ACcurrent, voltage, and/or frequency can be adjusted, for example, to heatthe fluid without substantially moving the fluid. Alternatively, powersupply 403 applies a pulse or impulse of current and/or voltage, whichpasses through channel 421 and into channel region 407 to heat fluid inregion 407 at a given instance in time. This pulse can be selectivelyadjusted to complement any voltage or electric field that may be appliedbetween regions 409 and 411 to move materials, e.g., fluids or othermaterials, in and out of region 407. Pulse width, shape, and/orintensity can be adjusted, for example, to heat the fluid substantiallywithout moving the fluids or materials, or to heat the material whilemoving the fluid or materials. Still further, the power supply may applyany combination of DC, AC, and pulse, depending upon the application.

[0141] A controller or computer 437 such as a personal computer,commonly termed PC, monitors the temperature of the fluid in region 407of the channel. The controller or computer receives current and voltageinformation from, for example, the power supply and identifies ordetects temperature of fluid in region 407 in the channel. Dependingupon the desired temperature of fluid in region 407, controller orcomputer adjusts voltage and/or current to meet the desired fluidtemperature. The controller or computer also can be set to be “currentcontrolled” or “voltage controlled” or “power controlled” depending uponthe application. Controller or computer 437 includes a monitor 439,which is often a cathode ray tube (“CRT”) display, a flat panel display(e.g., active matrix liquid crystal display, liquid crystal display),and others. Computer circuitry is often placed in a box 441, whichincludes numerous integrated circuit chips, such as a microprocessor,memory, interface circuits, and others. The box 441 also includes a harddisk drive, a floppy disk drive, a high capacity drive (e.g.,ZipDrive.TM. sold by Iomega Corporation), and other elements. Also shownare keyboard 443 and mouse 445, which provide for a human interface tocomputer box 441. A variety of techniques by way of a computer programcan be used to detect and monitor temperature, as well as other processparameters. Some of these techniques are described in more detail below.

[0142] In some embodiments, computer 437 is coupled to a network such asa local or wide area network. The local network can be configured as,for example, Ethernet or Token Ring. The local area network can also bean “Intranet.” Any one or a combination of these local area networks canbe connected to a wide area network such as the “Internet” among others.The network can also be wireless, depending upon the application. Thenetwork allows for users to be off-site or allows multiple users tomonitor or control or view processes of the present microfluidic system.

[0143] The embodiment shown in FIG. 7, for example, provides a higherfluid or material temperature in the channel at region 407 than atperipheral regions 421. Fluid in channel region 421 is maintained attemperature T0. Fluid in channel region 407 is maintained at temperatureTs The fluid temperature at region 407 is higher than the fluidtemperature at region 421 as a result of the higher current density (andhigher resistance) at region 407 from the cross-section of the channelat region 407 being smaller relative to the cross-section of the channelin region 421. Depending upon the shape of the channel, the temperatureprofile from one end of the channel to the other end of the channel canvary selectively. As can be appreciated, temperature control along thelength of the channel can be varied by varying the cross-sectionaldimension of that channel, while allowing the current to remainunchanged.

[0144] Example Geometric Variations

[0145]FIG. 8 is a simplified diagram of a microfluidic system with aheating source according to an alternative embodiment of the presentinvention. FIG. 7 illustrates, as one example, a simplified diagram of amicrofluidic system 500 with a heating source according to analternative embodiment of the present invention. This diagram is merelyan illustration and should not limit the scope of the claims herein. Oneof ordinary skill in the art would recognize other variations,alternatives, and modifications. The microfluidic system 500 includes achannel 502 having a variety of temperature zones, e.g., A, B, C, D, E,and fluid therein.

[0146] A first power supply, such as the one described above, provideselectric current between regions 501 and 503 for the purpose of heatingfluid in region 505, 509 and 513 of the channel configuration. The powersupply also provides a voltage differential between regions 501 and 503that drives the transport of material through channel 502. The electriccurrent is used, at least in part, to distribute energy to fluid inregion 509 for at least heating purposes. The power supply (or anotherpower supply) can also drive or move fluid between regions 501 and 503.A second signal source provides a probe signal between 501 and 503 asdescribed herein.

[0147] Channel 502 includes one type of geometric configuration, whichis designed to heat the fluid in region 505, 509 and 513 in an efficientmanner. As shown, channel 502 includes outer portions 507, 511 that eachhas a larger width or cross-section dimension than inner portion orregion 509. Additionally, regions 505 and 513 that attach, respectively,to regions 501 and 503, each has a smaller width or cross-sectiondimension than outer portions 507 and 511. The narrower dimensionsresult in an increased current density within these regions when acurrent is passed through channel 502, resulting in a heating of thefluid located within these regions. Accordingly, fluid also increases intemperature in regions 505 and 513. Material can also be transportedfrom region 501 to 503 while being heated in region 509, allowingheating of only a portion of the material. Further, additional regionsof narrower dimension are optionally provided along the length ofchannel 502, to provide thermal cycling, “on the fly,” as material istransported along channel 502. Other geometric configurations arediscussed in the cited references and these can also be used with aheating system and/or method according to specific embodiments of thepresent invention.

[0148] In a specific embodiment, fluid is heated in certain regions andcools in other regions. In particular, fluid in regions 507 and 511 iscooler than fluid in regions 505, 509, and 513. Additionally, channel502 can be coupled to other channels, which move fluid from one regionof channel 502 to another region of channel 502. Still further, thepower supplied between regions 501 and 503 can be varied depending uponthe application. For instance, the power source supplies energy in theform of electric current and/or voltage across regions 501 and 503. Theelectric current and/or voltage can be DC, AC, pulsed, a combinationthereof, and others. Of course, the type of power used depends upon theapplication.

[0149] A variety of methods can be used to globally raise or lower fluidtemperature in the microfluidic system, using energy sources or sinks toaffect this temperature change. These methods are described in citedreferences.

[0150] Numerous techniques can be used to control power to themicrochannels for the purpose of moving the fluid. These techniques canalso be used to selectively monitor and adjust temperature in themicrochannels or annular regions, as well. As noted above, one exampleof one of these techniques is the use of a processor or controllerand/or computer software such as the one described above. Alternatively,exclusively hardware or preferably a combination of hardware andsoftware can perform these techniques. Details with regard to specificcomputer programs that can perform selected techniques according to thepresent inventions are described below.

[0151] In a specific embodiment, the present invention provides atechnique used to detect and control temperature of a fluid being heatedin a microchannel by way of current and/or voltage and/or impedancemeasurements. Depending upon the application, other techniques may alsobe used. These techniques, of course, depend upon the application. Thepresent technique may be briefly described by way of the followingsequence of steps:

[0152] (1) Flow fluid in channel of microfluidic system;

[0153] (2) Stop flow of fluid in microchannel (optional);

[0154] (3) Apply first electric signal through conducting path to heatfluid;

[0155] (3) Apply probe electric signal through conducting path to probetemperature of fluid;

[0156] (4) Measure parameters of probe electric signal;

[0157] (5) Calculate temperature from probe electric signal;

[0158] (6) Compare actual temperature based upon a desired temperatureset-point;

[0159] (8) Adjust first electric signal applied based upon a differencebetween the actual temperature and the desired temperature set-point.

[0160] The above sequence of steps can be performed using, for example,a computer program. The computer program provides an easy-to-use methodto perform the above steps in a microfluidic system. The computerprogram can be executed in the form of computer software, firmware,hardware, or combinations thereof. The program executes the abovefunctions using an interface that is coupled to the microfluidic system.The interface receives signals from the microfluidic system and providessignals to, for example, the power source, which supplies electriccurrent to fluid for heating purposes.

[0161] In a modification to the preceding embodiment, the global oroverall temperature of the microfluidic system can be raised or loweredduring any one of the above process steps. Overall fluid temperature ispreferably globally raised or lowered using the technique describedabove, but can be others. Accordingly, fluid can be moved from oneregion to another in the microfluidic system. Fluid movement can becombined with selective heating of the fluid at a selected portion of amicrochannel and/or global fluid heating of the entire microfluidicsystem. Additionally, the fluid in the microfluidic system can be staticand heated globally or selectively in a specific location of themicrofluidic system.

[0162] The embodiments directed to controlling temperature of fluid canbe further modified or controlled by way of an active feedback processor control, using the probe signal, and depending upon the application.The active feedback process generally receives a signal such astemperature from the microfluidic process, for example. The measuredtemperature is compared with a set-point temperature. A difference iscalculated, taking into account any effects necessary for specificapplications, such as non-linear relationships between temperature andimpedance or additional factors needed for heat conductance when theelectrically conductive channel is not in direct contact with thesample. If the measured temperature is less than the set-point, a resultbased upon a function of the temperature difference is used to controladditional current or voltage to the fluid for heating purposes. Inpreferred embodiments, the function prevents any substantial “overshoot”or “oscillation” of the actual temperature from the set-pointtemperature. Additionally, the function ensures that the set-point isachieved in an efficient manner. Examples of functions used to providefeedback control include among others, proportional control,differential control, integral control, or a combination thereof.

[0163] Using, for example, proportional control, a feedback processaccording to the present invention provides an active feedback to theprocess based upon a multiplier. An output of a proportional controlleris a fixed multiple of a measured difference or “error.” That is, theproportional controller is simply the multiplier. Terms often used indescribing proportional controllers include a proportional band and acontroller gain. Controller gain is an amount by which the error ismultiplied to obtain an output. The controllers can be calibrated to theproportional band rather than the gain depending upon the application.

[0164] In a modification to the preceding embodiment, the global oroverall temperature of the microfluidic system can be raised or loweredduring any one of the above process steps. Overall fluid temperature ispreferably globally raised or lowered using the technique describedabove, but can be others. Accordingly, fluid can be moved from oneregion to another in the microfluidic system. Fluid movement can becombined with selective heating of the fluid in at a selected portion ofa microchannel and/or global fluid heating of the entire microfluidicsystem. Additionally, the fluid in the microfluidic system can be staticand heated globally or selectively in a specific location of themicrofluidic system.

[0165] Although descriptions herein are generally in terms of flowdiagrams, which can be carried out with computer software, the presentinventions can also be carried out in many other ways. For instance, thecomputer software can be placed in hardware such as a memory device,e.g., field programmable gate arrays (“FPGAs”), electrically erasableprogrammable read only memories (“EEPROMs”), read only memories(“ROMs”), random access memories (“RAMs”), etc. Another type of memorydevice would be a compact disk read only memories (“CDROMs”), harddisks, floppy disks, high capacity disks (e.g., ZipDrive.TM. sold byIomega Corporation), and others. Alternatively, the computer softwarecan be placed in a combination of hardware and software. Some of thefunctions described can be separated, or even combined, depending uponthe application. Of course, one of ordinary skill in the art wouldrecognize other variations, modifications, and alternatives, dependingupon the particular application.

[0166] Various applications of microfluidic devices according tospecific embodiments of the present invention will be readily understoodfrom the examples provided in the cited references. For example, variousprocesses for Amplifying and Detecting Nucleic Acids are described ine.g., U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; PCRTechnology: Principles and Applications for DNA Amplification (ed.Erlich, Freeman Press, New York, N.Y., 1992); PCR Protocols: A Guide toMethods and Applications (eds. Innis et al., Academic Press, San Diego,Calif. (1990); Mattila et al. Nucleic Acids Res. 19:4967 (1991); Eckert& Kunkel PCR Methods and Applications 1:17 (1991); PCR (eds. McPhersonet al., IRL Press, Oxford) (each of which is incorporated by referencein its entirety for all purposes). Reagents, apparatus, and instructionsfor using the same are commercially available. Other amplificationsystems include the ligase chain reaction, QB RNA replicase andRAN-transcription-based amplification systems.

[0167] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. For example, all the techniques described abovemay be used in various combinations. All publications and patentdocuments cited in this application are incorporated herein by referencein their entirety for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.

[0168] 9. Embodiment in a Programmed Information Appliance

[0169]FIG. 9 is a block diagram showing a representative example logicdevice in which various aspects of the present invention may beembodied. As will be understood to practitioners in the art from theteachings provided herein, the invention can be implemented in hardwareand/or software. In some embodiments of the invention, different aspectsof the invention can be implemented in either client-side logic orserver-side logic. As will be understood in the art, the invention orcomponents thereof may be embodied in a fixed media program componentcontaining logic instructions and/or data that when loaded into anappropriately configured computing device cause that device to performaccording to the invention. As will be understood in the art, a fixedmedia containing logic instructions may be delivered to a viewer on afixed media for physically loading into a viewer's computer or a fixedmedia containing logic instructions may reside on a remote server that aviewer accesses through a communication medium in order to download aprogram component.

[0170]FIG. 9 shows an information appliance (or digital device) 700 thatmay be understood as a logical apparatus that can read instructions frommedia 717 and/or network port 719, which can optionally be connected toserver 720 having fixed media 722. Apparatus 700 can thereafter usethose instructions to direct server or client logic, as understood inthe art, to embody aspects of the invention. One type of logicalapparatus that may embody the invention is a computer system asillustrated in 700, containing CPU 707, optional input devices 709 and711, disk drives 715 and optional monitor 705. Fixed media 717, or fixedmedia 722 over port 719, may be used to program such a system and mayrepresent a disk-type optical or magnetic media, magnetic tape, solidstate dynamic or static memory, etc. In specific embodiments, theinvention may be embodied in whole or in part as software recorded onthis fixed media. Communication port 719 may also be used to initiallyreceive instructions that are used to program such a system and mayrepresent any type of communication connection.

[0171] The invention also may be embodied in whole or in part within thecircuitry of an application specific integrated circuit (ASIC) or aprogrammable logic device (PLD). In such a case, the invention may beembodied in a computer understandable descriptor language, which may beused to create an ASIC, or PLD that operates as herein described.

[0172] 10. Other Embodiments

[0173] The invention has now been described with reference to specificembodiments. Other embodiments will be apparent to those of skill in theart. In particular, a viewer digital information appliance has generallybeen illustrated as a personal computer. However, the digital computingdevice is meant to be any information appliance for interacting with aremote data application, and could include such devices as a digitallyenabled television, cell phone, personal digital assistant, etc. Thus,although the present invention has been described in terms of variousspecific embodiments, it is not intended that the invention be limitedto these embodiments. Modification within the spirit of the inventionwill be apparent to those skilled in the art. It is understood that theexamples and embodiments described herein are for illustrative purposesand that various modifications or changes in light thereof will besuggested by the teachings herein to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the claims.

[0174] All publications, patents, and patent applications cited hereinor filed with this application, including any references filed as partof an Information Disclosure Statement, are incorporated by reference intheir entirety.

What is claimed:
 1. A method of controllably providing heating in amicrofluidic device, comprising: applying a first selectable currentthrough a resistive heating element in thermal contact with themicrofluidic device; applying a second selectable current through theresistive heating element, wherein the second selectable current has adifferent frequency than the first selectable current, detecting atleast one characteristic indicative of a temperature of the resistiveheating element using the second selectable current; and using the onecharacteristic to control the selectable current to elevate thetemperature of the resistive heating element.
 2. The method of claim 1,further wherein: the first selectable current comprises a higherfrequency signal; and the second selectable current comprises a lowerfrequency signal.
 3. The method of claim 1, further wherein: the firstselectable current comprises a higher frequency signal of about 10 kHz;and the second selectable current comprises a lower frequency signal ofabout 10 Hz.
 4. The method of claim 1, further wherein: the secondselectable current is used to measure the resistance of the resistiveheating element.
 5. The method of claim 1, further wherein: the firstselectable current comprises a frequency signal of greater than about300 Hz.
 6. The method of claim 1, wherein the resistive heating elementis disposed in a channel in the microfluidic device.
 7. The method ofclaim 1, wherein the resistive heating element is disposed near achannel in the microfluidic device.
 8. The method of claim 1, comprisingrepeatedly cycling a temperature of a material in a channel of themicrofluidic device between a first temperature and a secondtemperature.
 9. The method of claim 8, wherein the material comprisesreagents for performing a nucleic acid amplification reaction.
 10. Themethod of claim 9, wherein the nucleic acid amplification reaction isselected from the group consisting of a polymerase chain reaction and aligase chain reaction.
 11. The method of claim 1, wherein the firstselectable current comprises an alternating current and the secondselectable current comprises a direct current.
 12. The method of claim1, wherein the first selectable current comprises a direct current andthe second selectable current comprises an alternating current.
 13. Themethod of claim 1, wherein the microfluidic device comprises afluid-filled channel, and further comprising the step of maintaining aglobal temperature of the microfluidic device at a selected level aboveor below ambient temperature.
 14. A system for elevating temperature inat least a portion of a fluid-filled channel disposed in a substrate, toa selected elevated temperature, comprising: a resistive heating elementdisposed on the substrate; a controllable effector power source able toapply a first controllable signal through a fluid in the at least aportion of the fluid-filled channel; a probe signal source able to applya second voltage signal through a fluid in the at least a portion of thefluid-filled channel, wherein the second voltage signal has a differentfrequency than the first controllable signal; a probe signal detectorable to detect at least one characteristic indicative of a fluidtemperature using said probe signal; and a controller able to use saidat least one characteristic to provide a control signal varying saidcontrollable effector power source.
 15. The system of claim 14, furtherwherein: the first controllable signal comprises a higher frequency,higher voltage signal; and the probe signal comprises a lower frequency,lower voltage signal.
 16. The system of claim 14, wherein the firstcontrollable signal comprises a higher frequency signal of about 10 kHz;and the probe signal comprises a lower frequency signal of about 10 Hz.17. The system of claim 14, wherein the probe signal is used to measureresistance of the resistive heating element.
 18. The system of claim 14,wherein the first controllable signal comprises a frequency signal ofgreater than about 300 Hz.
 19. The system of claim 14, wherein theresistive heating element is disposed in the fluid filled channel. 20.The system of claim 14, wherein the resistive heating element isdisposed near the fluid filled channel.